Prominin‐1 expression in the testis/epididymis and fertility

Abstract The contribution of Prominin‐1 (aka CD133) to male fertility has recently been (re)investigated, with contradictory results. Early findings, essential for deciphering its role, have unfortunately been neglected. Here, the authors present what is currently known about its expression in the male reproductive system of rodents and men so that its involvement in male fertility can be re‐examined and discussed in the light of these elements.


| INTRODUC TI ON
Matsukuma and colleagues have recently reported that Prominin-1 deletion results in spermatogenic impairment, sperm morphological defects, and infertility in mice. 1 Prominin-1 (alias CD133) has been, for more than two decades, the subject of intensive research in various organ systems due to its expression in stem (cancer stem) cells.This pentaspan transmembrane glycoprotein (≈120 kDa) localizes specifically in highly curved membranes as in microvilli and cilia of diverse epithelial cells and in other types of protrusions of nonepithelial cells.It regulates the organization and/or dynamics of such membrane protrusions via its interactions with membrane lipids and cytoplasmic proteins.

| E XPRE SS I ON OF PROMININ -1 IN MALE REPRODUC TIVE SYS TEMS
We reported, in 2004, its expression in murine testes and epididymides. 2Using immunohistochemistry and electron microscopy, we demonstrated that Prominin-1 was concentrated in the stereocilia of the apical surface of principal cells lining the epididymal epithelium, all along the duct with the exception of the initial segment (see Table 1).Prominin-1 was also found on the tails of developing spermatozoa associated with contorted testis seminiferous tubules, suggesting that this molecule may play a role in spermiogenesis (the final stage of spermatogenesis), in line with its preferential subcellular localization in membrane protrusions. 2Several splice variants of Prominin-1 with alternative C-termini were characterized, exhibiting lower molecular weights and differential glycosylation compared with the 120-kDa kidney form originally identified.This means that, depending on the antibody used, the full spectrum of Prominin-1 variants may not be detected, notably in the reproductive system (Table 1). 2 Prominin-1 variants with different molecular weights are indeed differentially expressed in ductuli efferentes and the epididymal tract, 2 and only a 100-kDa form of Prominin-1 harboring a truncated cytoplasmic C-terminus appears in the testis. 2,3The relation of these alternative C-termini with differential function of Prominin-1 in these tissues remains to be experimentally addressed.
Prominin-1 transcripts were later mapped in the luminal zone of the contorted seminiferous tubules containing spermatids and in the epididymal duct by in situ hybridization (Table 1). 4  these findings and also failed to detect Prominin-1 protein in developing spermatozoa. 1The reason for these discrepancies was not discussed by the authors and remains to be determined.The use of an anti-Prominin-1 antibody (HPA004922; Sigma-Aldrich), which is directed against a sequence of human Prominin-1 sharing only 56% identity with its mouse counterpart, may be part of the answer.Actually, most anti-Prominin-1 antibodies do not cross-react between mouse and human counterparts. 5As this antibody is not reported to react with species other than human, its cross-reactivity with murine Prominin-1 needs to be demonstrated first. 1 As for the antibody (18470-1-AP, Proteintech) used for the immunoblot of testicular samples, 1 although presented as reactive against mouse Prominin-1 despite being also raised against human Prominin-1, it is specifically directed against the C-terminal region of the 120-kDa kidney form that is partly absent in the 100-kDa variant expressed in the testis (Table 1).It would therefore be advisable to run samples (kidney and testis) in parallel and probe them with two different antibodies to identify the variants expressed.
Mammalian spermatozoa gain fertility potential during their transit through the epididymis.In such context, Prominin-1 may participate in sperm maturation, either by the generation of small vesicles conveying maturation factors from the epididymis, as seminal fluid does contain Prominin-1 + extracellular vesicles, or through its shedding from spermatozoa with other sperm constituents.Indeed, Asano and colleagues suggested that murine sperm cells acquired Prominin-1 in the testis, and not gradually during epididymal maturation (Table 1). 6ngruently, Prominin-1 was hardly detected on rat spermatozoa in the luminal compartment of the ductuli efferentes, initial segment, TA B L E 2 Prom1-ablated murine models.and caput, and somewhat inconsistently, in the corpus epididymis. 7 humans, Prominin-1 was detected in sperm cells, epididymal epithelium, 8,9 and sporadically in fetal tissues in the second but not the third trimester of gestation (Table 1). 8Consistent with its expression in germ cells, upregulation of Prominin-1 was observed in seminoma.
In normal adult testicular tissues, its mRNA expression was nonetheless hardly detectable by PCR. 8While evaluating differences in expression profiles of biomarkers between healthy and infertile men, Yukselten and colleagues have shown that Prominin-1 is expressed in healthy Sertoli cells and sperm cells (Table 1). 9How these differences between species are related to the expression of distinct Prominin-1 variants, to differential regulation or simply reflect a constitutive interspecific divergence might be worth exploring.

| D IFFERENT MURINE MODEL S OF PROMININ -1 DEFICIEN C Y
Matsukuma and colleagues also report that deletion of Prominin-1 results in complete infertility in male mice, 1 which seems to contradict other reports of diverse homozygous Prominin-1-deficient mouse models being viable and fertile (Table 2).These animal models show various retinal degeneration phenotypes, but no apparent fertility problems.Of note, Karim and colleagues were the first to observe compromised spermatogenesis in some (without indicating in which proportion) Prom1 −/− males on a less characterized 129SvEv background, although they reported no interference with development or fertility in general. 10It appears therefore that the drastic effect on male fertility reported by Matsukuma and colleagues may be influenced by factors other than the sole absence of Prominin-1. 1 One cannot exclude that a substrain of Prom1 −/− may have been derived given that the original publication in 2009 of these engineered animals did not report fertility issues and the distributor (Strain Data Sheet, BRC No. RBRC05284; RIKEN BRC, Japan) describes homozygous mice as viable and fertile.
Difficulties in breeding on the C57BL6 background were reported in 2015 and remedied by generating a hybrid C57BL/6xCBA/NSlc knockout through in vitro fertilization (Table 2).The differences in phenotype of the different knockout models, whether due to environmental factors or related to the strains and backgrounds of the mice, call for more detailed studies with established tools and larger cohorts of samples, to clarify the involvement of Prominin-1 in spermatogenesis.In particular, it could be interesting to see whether the other Prominin-1-deficient mouse models develop in a time course study the same tendency to higher sperm defect, to pinpoint the role of Prominin-1 in male fertility.
Finally, it is noteworthy that, although numerous recessive and dominant mutations in human PROM1 gene were reported to cause retinal degeneration, no other major phenotype, including infertility, was, to our knowledge, documented in these patients.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

A N I M A L R I G HTS
No animal experiments have been performed in this article.

R E FE R E N C E S
Matsukuma and   colleagues confirmed this last observation by β-galactosidase staining of heterozygote animals of a knock-in mouse model carrying the LacZ gene in the Prom1 locus.1 Strikingly, while neither Prominin-1 transcript expression4 nor LacZ staining 1 were detected in the basal portion of seminiferous tubules containing actively dividing spermatogonia, the immunodetection in the latter study contradicted TA B L E 1 Detection of Prominin-1 in testis and epididymis.presence of Prominin-1; −, absence of Prominin-1; grayed, not applicable or not determined.Abbreviations: HC, histochemistry; WB, western blot.a 13A4, rat monoclonal antibody (mAb) targeting the second extracellular domain of mouse Prominin-1, recognizes all splice variants in testis/epididymis.b αI3, rabbit antiserum targeting the C-terminal domain of mouse Prominin-1.s1, recognizes only non-truncated variants.c Antiserum against Prominin-1 from eBioscience, not specified.d 18 470-1-AP from Proteintech, antigen-purified rabbit polyclonal targeting the C-terminus (amino acid 806-856) of human Prominin-1.s1,reacts with human, mouse, and rat; recognition of splice variants affecting the C-terminal domain has not been validated.e HPA004922 from Sigma-Aldrich, antigen-purified rabbit polyclonal targeting sequence in the 2nd extracellular domain (amino acid 209-315) of human Prominin-1.s1,reacts with human, not validated for mouse.f β-Galactosidase staining on Prom1 +/lacZ,DTA .g ISH, in situ hybridization of Prominin-1 transcript.h Mouse anti-PROM1 mAb from ABGENT, not specified.i Anti-CD133 mAb from Miltenyi Biotec, not specified.j Molecular weights not precisely determined, but consistent with those of Ref. 2 using the same antibody.k 4 or more immunoreactive bands up to 150 kDa present in Prom1 +/+ and Prom1 −/− testis.l Results obtained on micro-testicular sperm extraction of obstructive azoospermia patients show additional bands of lower molecular weight.m Parentheses indicate elements visible on the figure, but not mentioned as such by the authors.